Method for preparing cell extract component or composition having cytocidal activity

ABSTRACT

Provided is a method or the like for producing a composition exhibiting cytocidal activity. This method for producing a composition exhibiting cytocidal activity comprises: culturing malignant tumor-derived cells in a culture medium at least until the cell density reaches a level that does not pose a problem for transfer; replacing, after culturing, the culture medium with a physiological buffer salt solution; and recovering the physiological buffer salt solution after death of the malignant tumor-derived cells is observed morphologically in the physiological buffer salt solution.

TECHNICAL FIELD

The present invention relates to a method for producing a compositionhaving cytocidal activity, a method for preparing a cell extractcomponent having cytocidal activity, and the like.

BACKGROUND ART

When cells are cultured, many established cell lines can growindefinitely if they are dispersed with trypsin or the like at the timewhen the cells have grown up and have covered fully the culture vesselsurface, diluted in fresh medium, and transferred to another culturevessel for culturing. This is called passage, and if culturing iscontinued without passage, the cells die.

Patent Literature 1 discloses a malignant tumor cell growth inhibitorobtained by removing malignant tumor cells from a medium after culturingmalignant tumor cells. However, what was obtained by removing malignanttumor cells from the medium was a composition containing extremelymiscellaneous substances, and the isolation of substances havingantineoplastic activity from such a composition was greatly difficultand considered to be virtually impossible.

CITATION LIST Patent Literatures

Patent Literature 1: Japanese Patent Application Publication No. Sho59-33223

SUMMARY OF INVENTION

To date, various therapeutic agents have been researched and developedfor the purpose of cancer treatment. However, since conventionalanticancer agents often have strong side effects and fail to achievesufficient effects, there is still a need for further drug development.

An aspect of the present invention makes it possible to provide a methodfor producing, from cultured cells, a composition which can be used as anovel anticancer agent, or a composition which can be used to obtain asubstance useful as a novel anticancer agent.

In addition, an aspect of the present invention can provide a methodwhich makes it possible to obtain a cell extract component havingcytocidal activity.

The present inventors have made earnest studies and have newly foundthat the phenomenon of cell death in the case of continuing cell culturewithout passage appears even if the cells are replenished withsufficient nutrients and energy sources by medium replacement at thetime when growth suppression occurs due to overpopulation after thecells cover fully the culture vessel surface to further grow.

In addition, the present inventors have newly found that malignanttumor-derived cells produce a substance which causes themselves to dieeven in the case where the cells are cultured in a culture medium atleast until a cell density reaches a level that does not pose anobstacle for passage, and then the culture medium is replaced with aphysiological buffer salt solution (containing no nutrient and/or energysource). This is a finding indicating that cells produce a substancehaving cytocidal activity from only substances present in the cells, andcause themselves to die. Such finding has not been reported or suggestedso far, and it is utterly unexpected that a physiological buffer saltsolution can be used to obtain a component having cytocidal activityextracted from malignant tumor-derived cells.

An embodiment of the present invention relates to the following.

[1] A method for producing a composition having cytocidal activity, themethod comprising:

culturing malignant tumor-derived cells in a culture medium at leastuntil a cell density reaches a level that does not pose an obstacle forpassage;

replacing, after the culturing, the culture medium with a physiologicalbuffer salt solution; and

recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor-derived cells is observed in thephysiological buffer salt solution in morphological aspect of saidcells.

[2] The production method according to [1] described above, wherein thephysiological buffer salt solution is glucose-free.

[3] The production method according to [1] or [2] described above,wherein the physiological buffer salt solution is selected from thegroup consisting of Hanks' balanced salt solution, Earle's balanced saltsolution, and phosphate buffered saline.

[4] The production method according to any one of [1] to [3] describedabove, further comprising

obtaining a dried product containing a fraction with a molecular weightof 1 kDa or less in the recovered physiological buffer salt solution,and

dissolving the dried product in a medium to obtain a solution andremoving a salt, a nucleic acid, and a protein from the resultingsolution.

[5] The production method according to any one of [1] to [4] describedabove, wherein the composition having cytocidal activity contains a cellextract component derived from the malignant tumor-derived cells.

[6] A method for preparing a cell extract component derived frommalignant tumor-derived cells, the method comprising:

culturing malignant tumor-derived cells in a culture medium at leastuntil a cell density reaches a level that does not pose an obstacle forpassage;

replacing, after the culturing, the culture medium with a physiologicalbuffer salt solution;

recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor-derived cells is observed in thephysiological buffer salt solution in morphological aspect of saidcells;

obtaining a dried product containing a fraction with a molecular weightof 1 kDa or less in the recovered physiological buffer salt solution;

extracting the dried product using a solvent containing an alcoholhaving 1 to 3 carbon atoms, and drying a resultant solution;

dissolving a dried product of the resultant solution in water to obtainan aqueous solution, adding a non-polar organic solvent to the aqueoussolution to form an aqueous layer and an organic layer, and extractingthe aqueous layer; and

separating a cell extract component derived from the malignanttumor-derived cells from the aqueous layer by chromatography, wherein

the cell extract component has cytocidal activity.

[7] The preparation method according to [6] described above, wherein thechromatography includes gel filtration chromatography and/or cationexchange chromatography.

[8] The production method or the preparation method according to any oneof [1] to [7] described above, wherein the malignant tumor-derived cellsare not genetically engineered and are cultured without adding aphysiologically active substance other than a culture solution.[9] A composition having cytocidal activity which is obtained by theproduction method according to any one of [1] to [5] and [8] describedabove.[10] A cell extract component derived from malignant tumor-derived cellswhich is obtained by the preparation method according to [6] or [7]described above.[11] A pharmaceutical composition for treating cancer comprising thecomposition having cytocidal activity obtained by the production methodaccording to any one of [1] to [5] and [8] described above.[12] A pharmaceutical composition for treating cancer comprising, as anactive ingredient, the cell extract component derived from the malignanttumor-derived cells obtained by the preparation method according to [6]or [7] described above.[13] Use of the composition having cytocidal activity obtained by theproduction method according to any one of [1] to [5] and [8] describedabove for the production of a medicament for treating cancer.[14] Use of the cell extract component derived from the malignanttumor-derived cells obtained by the preparation method according to [6]or [7] described above for the production of a medicament for treatingcancer.

An aspect of the present invention makes it possible to inexpensively,conveniently, and/or in a short period of time, produce a compositionwhich can be used as a novel anticancer agent, or a composition whichcan be used to obtain a substance useful as a novel anticancer agent.

An aspect of the present invention makes it possible to obtain a cellextract component having cytocidal activity inexpensively, conveniently,and/or in a short period of time. The cell extract having cytocidalactivity may be effective against various cancers.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (acolorimetric assay for assessing cell metabolic activity) in a serialdilution series of test samples prepared from HRC23 using aserum-containing medium (Eagle's MEM with 10% FBS). The horizontal axisrepresents the concentration of the test sample (undiluted solutionmL/mL) on a common logarithmic scale.

FIG. 2 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from HRC23 using a serum-freemedium (Eagle's MEM). “MEM≤1 kDa” is the fraction with a molecularweight of 1 kDa or less of the sample prepared using the Eagle's MEM.The horizontal axis represents the concentration of the test sample(undiluted solution mL/mL) on a common logarithmic scale.

FIG. 3 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from HRC23 using aphysiological buffer salt solution (glucose-free Hanks' balanced saltsolution: HBSS−). “HBSS−≤1 kDa” is the fraction with a molecular weightof 1 kDa or less of the sample prepared using the HBSS−. The horizontalaxis represents the concentration of the test sample (undiluted solutionmL/mL) on a common logarithmic scale.

FIG. 4 is a chromatogram by gel filtration chromatography. Of the eightfractions A to H, the fraction A in which cytocidal activity wasdetected (105 to 141 minutes) is indicated with hatching.

FIG. 5 illustrates the appearance of color development on a microplateobserved by MTT assay in a serial dilution series for the fraction Aobtained by gel filtration chromatography, together with a semi-loggraph illustrating the average value and standard deviation of themeasured cell survival rate. The horizontal axis represents theconcentration of the test sample (undiluted solution mL/mL) on a commonlogarithmic scale.

FIG. 6 is a chromatogram by ion exchange chromatography, and illustrateswith hatching the fractions in which cytocidal activity was detected(fractions with Na₂SO₄ concentration of 165 to 170 mM).

FIG. 7 is a chromatogram by gel filtration chromatography. The cytocidalactivity is detected in the earlier peak, which includes three fractions(Fr57, Fr58, and Fr59).

FIG. 8 is a mass spectrum for fraction 57.

FIG. 9 is a mass spectrum for fraction 58.

FIG. 10 is a mass spectrum for fraction 59.

FIG. 11 is a mass spectrum for matrix only.

FIG. 12 is an enlarged diagram of the vicinity of signals at the m/zvalue of 114.09 in the spectrum of fraction 58.

FIG. 13 is a semi-log graph illustrating the average value and standarddeviation of cell survival rate of various cells measured by MTT assayin a serial dilution series of test samples prepared from HRC23 using aphysiological buffer salt solution (glucose-free Hanks' balanced saltsolution: HBSS−). The horizontal axis is represented on a commonlogarithmic scale, where the minimum concentration of the test sampleexhibiting 0% survival rate for HRC23 is expressed as 1 in arbitraryunit.

FIG. 14 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from LLC using a physiologicalbuffer salt solution (glucose-free Hanks' balanced salt solution:HBSS−). “Whole” refers to the whole sample and “≤1 kDa” refers to thefraction of the sample with a molecular weight of 1 kDa or less. Thehorizontal axis represents the concentration of the test sample(undiluted solution mL/mL) on a common logarithmic scale.

FIG. 15 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from SKN using a physiologicalbuffer salt solution (glucose-free Hanks' balanced salt solution:HBSS−). “Whole” refers to the whole sample and “≤1 kDa” refers to thefraction of the sample with a molecular weight of 1 kDa or less. Thehorizontal axis represents the concentration of the test sample(undiluted solution mL/mL) on a common logarithmic scale.

FIG. 16 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from HRC23 using aphysiological buffer salt solution (glucose-free Hanks' balanced saltsolution: HBSS−, Earle's balanced salt solution: Earle, and phosphatebuffered saline: PBS(+)). The horizontal axis represents theconcentration of the test sample (undiluted solution mL/mL) on a commonlogarithmic scale.

FIG. 17 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from HRC23 with different celldensities after culturing using a physiological buffer salt solution(glucose-free Hanks' balanced salt solution: HBSS−). The horizontal axisrepresents the concentration of the test sample (undiluted solutionmL/mL) on a common logarithmic scale.

FIG. 18 is a semi-log graph illustrating the average value and standarddeviation of LK-2 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from LK-2 using aserum-containing medium (RPMI 1640 with 10% FBS) or a physiologicalbuffer salt solution (glucose-free Hanks' balanced salt solution:HBSS−). “HBSS−(≤1 kDa)” is the fraction of the sample with a molecularweight of 1 kDa or less prepared using the glucose-free Hanks' balancedsalt solution (HBSS−). The horizontal axis represents the concentrationof the test sample (undiluted solution mL/mL) on a common logarithmicscale.

FIG. 19 is a semi-log graph illustrating the average value and standarddeviation of HRC23 cell survival rate measured by MTT assay in a serialdilution series of test samples prepared from LK-2 using aserum-containing medium (RPMI 1640 with 10% FBS) or a physiologicalbuffer salt solution (glucose-free Hanks' balanced salt solution:HBSS−). “HBSS− (≤1 kDa)” is the fraction of the sample with a molecularweight of 1 kDa or less prepared using the glucose-free Hanks' balancedsalt solution (HBSS−). The horizontal axis represents the concentrationof the test sample (undiluted solution mL/mL) on a common logarithmicscale.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention is described in detail.

An embodiment of the present invention is a method for producing acomposition having cytocidal activity, the method comprising:

culturing malignant tumor-derived cells in a culture medium at leastuntil a cell density reaches a level that does not pose an obstacle forpassage;

replacing, after the culturing, the culture medium with a physiologicalbuffer salt solution; and

recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor-derived cells is observed in thephysiological buffer salt solution in morphological aspect of saidcells.

In the present specification, the “malignant tumor-derived cell” means acultured cell (primary cultured cell) obtained from a malignant tumor ora malignant tumor-derived established cell line. Established culturedcell lines have a characteristic in common that they can be culturedindefinitely if subjected to passage, and die even in a fresh mediumwithout passage. From this, it is considered that a cell-derivedsubstance having cytocidal activity is involved in the “natural celldeath” (necrosis) to kill the cells. Therefore, all established celllines are considered to be a raw material for producing substanceshaving such cytocidal activity.

The “malignant tumor” is generally referred to as cancer and is used ina broad sense including carcinoma, sarcoma, and hematologic malignancy(hematopoietic tumor).

For example, whether epithelial or non-epithelial, the “malignanttumor-derived cells” may be derived from cancer such as lung cancer,gastric cancer, esophageal cancer, liver cancer, biliary tract cancer,pancreatic cancer, large bowel cancer, renal cancer, bladder cancer,prostatic cancer, testicular cancer, uterine cancer, ovarian cancer,breast cancer, skin cancer, laryngeal cancer, colorectal cancer,melanoma, thyroid cancer, fibrosarcoma, dermatofibrosarcoma, uterinesarcoma, liposarcoma, myosarcoma, hemangiosarcoma, Kaposi's sarcoma,lymphangiosarcoma, osteosarcoma, leukemia, lymphoma, and myeloma. Thesecancers may be derived from humans or may be derived from mammals(except for humans) such as mice.

In addition, whether epithelial or non-epithelial, the “malignanttumor-derived cells” are not limited to adenocarcinoma, squamous cellcarcinoma, small cell carcinoma, and large cell carcinoma, and may bederived from any tissue type including sarcoma.

In an embodiment of the present invention, the “malignant tumor-derivedcells” may be ones which are not genetically engineered and are culturedwithout adding a physiologically active substance other than a culturesolution.

In the production method of the present invention, the “culturingmalignant tumor-derived cells in a culture medium at least until a celldensity reaches a level that does not pose an obstacle for passage” canbe appropriately carried out based on known techniques.

For example, the culturing at least until a cell density reaches a levelthat does not pose an obstacle for passage can be culturing cells to aconfluent state, or culturing cells to a fully confluent state. Inaddition, the culturing at least until a cell density reaches a levelthat does not pose an obstacle for passage is not limited to the above,and may be culturing cells to a 60 to 100% confluent state, preferably a70 to 100% confluent state, may be culturing cells until the adherentsurface of the culture vessel is completely covered or until theadherent surface of the culture vessel is covered at least about 80% ifthe cells are adherent cells, or may be culturing cells until the liquidsurface of the culture medium is completely covered or until the liquidsurface of the culture medium is covered at least about 80% if the cellsare floating cells. From the viewpoint of the efficiency of producing acell extract component having cytocidal activity, it is more preferableto culture cells to a 80 to 100% confluent state, it is furtherpreferable to culture cells to a 90 to 100% confluent state, and it iseven more preferable to culture cells to a confluent state or a fullyconfluent state. Whether or not at least the density of cells hasreached a level that does not pose an obstacle for passage can beappropriately determined based on the ordinary knowledge of thoseskilled in the art.

In the production method of the present invention, the culture mediumused may be one which is suitable for the malignant tumor-derived cellsto be used. For example, the culture medium includes Eagle's MEM,Dulbecco's modified MEM, RPMI 1640, HAM F-12, a completely syntheticmedium which does not require FBS, and the like. In addition, ifnecessary, these culture media may be added with vitamins, coenzymes,amino acids, metal ions, sugars, cell growth factors, interleukins,cytokines, serum, serum-derived components, antibiotics, and the like.

In the production method of the present invention, the malignanttumor-derived cells used may already be subjected to passage, and thepassage can be conducted based on the ordinary knowledge of thoseskilled in the art.

In the production method of the present invention, the “replacing theculture medium with a physiological buffer salt solution” is, forexample, removing the culture medium from the culture vessel such as aculture flask and then adding a physiological buffer salt solution tothe culture vessel.

The physiological buffer salt solution is not particularly limited to,but includes, for example, Hanks' balanced salt solution (HBSS), Earle'sbalanced salt solution, phosphate buffered saline (PBS), Ringer'sbalanced salt solution, Simms' balanced salt solution, Tyrode's balancedsalt solution, Gey's balanced salt solution, Puck's balanced saltsolution, Eagle's balanced salt solution, and the like. Preferably, thephysiological buffer salt solution is glucose-free. An additionalcomponent such as an antibiotic may or may not be added to thephysiological buffer salt solution.

In an embodiment of the present invention, the physiological buffer saltsolution is Hanks' balanced salt solution, Earle's balanced saltsolution, or phosphate buffered saline, and is preferably glucose-free.More preferably, the physiological buffer salt solution is glucose-freeHanks' balanced salt solution (HBSS−).

In the production method of the present invention, the “recovering thephysiological buffer salt solution after the time at which death of themalignant tumor-derived cells is observed in the physiological buffersalt solution in morphological aspect of said cells” is, for example,recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor-derived cells can be confirmed inmorphological aspect of the cells by microscopic observation, MTTmethod, or the like. The observation or confirmation of morphologicalcell death of the malignant tumor-derived cells can be appropriatelydetermined based on the ordinary knowledge of those skilled in the artdepending on the type of malignant tumor-derived cells used. Forexample, if cells are seen peeled off when the flask is lightly hit orif cells (fragments) are seen suspended with the naked eye, it isrecognized that morphological cell death of the malignant tumor-derivedcells can be observed or confirmed.

The “after the time at which death of the malignant tumor-derived cellsis observed in morphological aspect of said cells” may vary depending onthe type of malignant tumor-derived cells used and the conditions ofcell culture, and may be, for example, three to seven days after the daywhen a culture medium is replaced with a physiological buffer saltsolution. Even if dead cell fragments are contained after recovery, theyare stable for 1 month at 4° C. under aseptic conditions at the levelduring culture.

In addition, it is preferable to centrifuge a physiological buffer saltsolution after recovery, and to collect a resultant supernatant. Theconditions for centrifugation may be, but are not limited to, 1,000 to17,000×g for 10 to 20 minutes at 4° C. to room temperature (for example,25° C.). In addition, it is preferable to filter the supernatant using amembrane filter such as a 0.1 μm membrane filter, and to collect afiltrate.

In the production method of the present invention, it is possible toappropriately select the conditions for culturing malignanttumor-derived cells in a physiological buffer salt solution after thetime at which death of the malignant tumor-derived cells is observed inmorphological aspect of the cells and until recovering the physiologicalbuffer salt solution. Basically, in the case of a physiological buffersalt solution designed to be an open system such as HBSS−, incubation iscarried out at 36° C. to 37° C. in an incubator or atemperature-controlled room in a closed system which does not allowwater evaporated in the atmosphere to escape. Alternatively, malignanttumor-derived cells may be incubated in a physiological buffer saltsolution under the same conditions as the ordinary culture conditions.For example, the same conditions as the ordinary culture conditions are,but not limited to, a temperature range of 30° C. to 38° C., preferably35° C. to 37° C., a humidity range of 70 to 100%, preferably 90 to 100%,and a carbon dioxide range of 2 to 8%, preferably 4 to 6%. In addition,a physiological buffer salt solution which requires pH control withcarbon dioxide gas can be used as well, with a reduced NaHCO₃ content.

The production method of the present invention may further includeobtaining a dried product containing a fraction with a molecular weightof 1 kDa or less in the recovered physiological buffer salt solution.The fraction with a molecular weight of 1 kDa or less can be obtained bya known means such as ultrafiltration using a commercially availablemembrane filter or the like. In addition, the dried product containing afraction with a molecular weight of 1 kDa or less can be obtained by aknown means such as vacuum drying.

The production method of the present invention may further includedissolving the thus obtained dried product in a medium to obtain asolution and removing a salt, a nucleic acid, and a protein from theresulting solution. Specifically, the dissolving the dried product in amedium to obtain a solution and removing a salt, a nucleic acid, and aprotein from the resulting solution can be carried out by, but notlimited to, the following steps.

(1) The step of extracting the dried product using a solvent containingan alcohol having 1 to 3 carbon atoms, and drying a resultant solution,and the step of dissolving a dried product of the resultant solution inwater to obtain an aqueous solution and adding an organic solvent to theaqueous solution or adding a dried product of the resultant solution toa water-added organic solvent to form an aqueous layer and an organiclayer, and extracting the aqueous layer.

Here, the solvent containing an alcohol having 1 to 3 carbon atoms isspecifically a solvent containing methanol, ethanol, n-propyl alcohol,or isopropyl alcohol. The solvent containing an alcohol having 1 to 3carbon atoms may be a liquid mixture with e.g. an alcohol or an organicsolvent such as chloroform.

In addition, the organic solvent is not particularly limited as long asit is sufficient to form an aqueous layer and an organic layer, andexamples thereof usable include non-polar organic solvents,specifically, chloroform, a liquid mixture of chloroform and ethylacetate, and the like.

The “extracting the dried product using a solvent containing an alcoholhaving 1 to 3 carbon atoms” means, for example, adding a solventcontaining an alcohol having 1 to 3 carbon atoms to a dried product,followed by centrifugation at 1000 to 2000×g for 5 to 10 minutes toobtain a supernatant.

(2) The step of adding the dried product to a water-added organicsolvent to form an aqueous layer and an organic layer, and extractingthe aqueous layer, and the step of removing a salt and the like from theaqueous layer by chromatography.

Here, the organic solvent is not particularly limited as long as it issufficient to form an aqueous layer and an organic layer. Although gelfiltration chromatography is preferable for removing a salt and the likeby chromatography, it is not limited thereto.

The composition having cytocidal activity obtained by the productionmethod of the present invention contains a cell extract component havingcytocidal activity derived from malignant tumor-derived cells. Asdemonstrated in Examples to be described later, the cell extractcomponent having cytocidal activity is effective against variouscancers, and even against mouse Lewis lung carcinoma, which isconsidered to have resistance to various anticancer agents.

A glucose-free physiological buffer salt solution is a medium which hasno nutrients and no energy sources for cells. Therefore, it has beenrevealed that the cell extract component having cytocidal activityderived from malignant tumor-derived cells, which is contained in thecomposition having cytocidal activity obtained by the production methodof the present invention, is produced by the cells using only thesubstances present in the cells as materials.

Serum-containing media and serum-free media used for culturing cellscontain various components suitable for culturing cells. Thus, it isextremely difficult to isolate and purify a cell extract componenthaving cytocidal activity derived from malignant tumor-derived cellsfrom serum-containing media and serum-free media. On the other hand,isolation and purification of a cell extract component having cytocidalactivity derived from malignant tumor-derived cells from a physiologicalbuffer salt solution is easier than the isolation and purification fromserum-containing media and serum-free media. Therefore, the productionmethod of the present invention can make it possible to obtain a cellextract component having cytocidal activity inexpensively, conveniently,and/or in a short period of time. In particular, consider the case ofusing a glucose-free physiological buffer salt solution. The compositionhaving cytocidal activity obtained by the production method of thepresent invention becomes a system which does not contain an energysource, making it possible to reduce the amount of lactic acid producedand to prevent a decrease in pH. In addition, since lactic acid issoluble in an organic solvent such as ethanol, reducing the amount oflactic acid produced makes it possible to suppress the influence onpurification.

An embodiment of the present invention is a method for preparing a cellextract component derived from malignant tumor-derived cells, the methodcomprising:

culturing malignant tumor-derived cells in a culture medium at leastuntil a cell density reaches a level that does not pose an obstacle forpassage;

replacing, after the culturing, the culture medium with a physiologicalbuffer salt solution;

recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor-derived cells is observed in thephysiological buffer salt solution in morphological aspect of saidcells;

obtaining a dried product containing a fraction with a molecular weightof 1 kDa or less in the recovered physiological buffer salt solution;

extracting the dried product using a solvent containing an alcoholhaving 1 to 3 carbon atoms, and drying a resultant solution;

dissolving a dried product of the resultant solution in water to obtainan aqueous solution, adding a non-polar organic solvent to the aqueoussolution to form an aqueous layer and an organic layer, and extractingthe aqueous layer; and

separating a cell extract component derived from the malignanttumor-derived cells from the aqueous layer by chromatography, wherein

the cell extract component has cytocidal activity.

In the preparation method of the present invention, the “culturingmalignant tumor-derived cells in a culture medium at least until a celldensity reaches a level that does not pose an obstacle for passage,” the“replacing the culture medium with a physiological buffer saltsolution,” the “recovering the physiological buffer salt solution afterthe time at which death of the malignant tumor-derived cells is observedin the physiological buffer salt solution in morphological aspect ofsaid cells,” and the “obtaining a dried product containing a fractionwith a molecular weight of 1 kDa or less in the recovered physiologicalbuffer salt solution” are the same as those in the above-describedmethod for producing a composition having cytocidal activity.

In the preparation method of the present invention, the “extracting thedried product using a solvent containing an alcohol having 1 to 3 carbonatoms, and drying a resultant solution” can be carried out by anordinary extraction operation.

Here, the solvent containing an alcohol having 1 to 3 carbon atoms isspecifically a solvent containing methanol, ethanol, n-propyl alcohol,or isopropyl alcohol. The solvent containing an alcohol having 1 to 3carbon atoms may be a liquid mixture with e.g. an alcohol or an organicsolvent such as chloroform.

The “extracting the dried product using a solvent containing an alcoholhaving 1 to 3 carbon atoms” means, for example, adding a solventcontaining an alcohol having 1 to 3 carbon atoms to a dried product,followed by centrifugation at 1000 to 2000×g for 5 to 10 minutes toobtain a supernatant.

In the preparation method of the present invention, the “dissolving adried product of the solution in water to obtain an aqueous solution,adding a non-polar organic solvent to the aqueous solution to form anaqueous layer and an organic layer, and extracting the aqueous layer”can be carried out by an ordinary extraction operation.

The non-polar organic solvent is not particularly limited as long as itis sufficient to form an aqueous layer and an organic layer, andexamples thereof usable include chloroform, a liquid mixture ofchloroform and ethyl acetate, and the like.

In the preparation method of the present invention, the “separating acell extract component derived from the malignant tumor-derived cellsfrom the aqueous layer by chromatography” can be carried out by a knownchromatography technique. Preferably, gel filtration chromatographyand/or cation exchange chromatography is used to separate cell extractcomponents having cytocidal activity derived from malignanttumor-derived cells.

Here, gel filtration chromatography and cation exchange chromatographycan be appropriately carried out using commercially availableapparatuses, carriers, and columns. The cation exchange chromatographymay elute the target substance by the gradient method (concentrationgradient method), or may elute the target substance by the isocraticmethod (isocratic elution method).

In cation exchange chromatography, it is preferable to use a strongcation exchange column.

An embodiment of the present invention relates to a composition havingcytocidal activity which is obtained by the above-described productionmethod of the present invention.

An embodiment of the present invention relates to a cell extractcomponent derived from malignant tumor-derived cells which is obtainedby the above-described preparation method of the present invention.

The composition having cytocidal activity, which is obtained by theabove-described production method of the present invention, is derivedfrom malignant tumor-derived cells, and it is impossible or impracticalto directly identify said composition with the structure orcharacteristics thereof. Regarding the cell extract component derivedfrom malignant tumor-derived cells obtained by the preparation method ofthe present invention, the structural identification of that substancerequires a large quantity of pure product samples and very expensivemeasuring equipment. In addition, it is necessary to examine variousproperties of the substance such as stability and then to repeat a greatnumber of trials and errors, which requires a great deal of time andmoney. Therefore, the structure identification is nearly impractical.

An embodiment of the present invention relates to a pharmaceuticalcomposition for treating cancer comprising the composition havingcytocidal activity obtained by the above-described production method ofthe present invention.

An embodiment of the present invention relates to a pharmaceuticalcomposition for treating cancer comprising, as an active ingredient, thecell extract component derived from the malignant tumor-derived cellsobtained by the above-described preparation method of the presentinvention.

An embodiment of the present invention relates to use of the compositionhaving cytocidal activity obtained by the above-described productionmethod of the present invention for the production of a medicament fortreating cancer.

An embodiment of the present invention relates to use of the cellextract component derived from the malignant tumor-derived cellsobtained by the above-described preparation method of the presentinvention for the production of a medicament for treating cancer.

Cancers which can be treated by the pharmaceutical composition fortreating cancer or the medicament for treating cancer of the presentinvention are not limited by the types of malignant tumor-derived cellsused in the above-described production method or preparation method ofthe present invention. Specifically, the pharmaceutical composition fortreating cancer or the medicament for treating cancer of the presentinvention can be effective against the same type of cancer as ordifferent types of cancer from malignant tumor-derived cells used in theabove-described production method or preparation method of the presentinvention.

In addition, the pharmaceutical composition for treating cancer or themedicament for treating cancer of the present invention can be effectivein any of carcinoma, sarcoma, and hematologic malignancy (hematopoietictumor). Cancers which can be treated by the pharmaceutical compositionfor treating cancer or the medicament for treating cancer of the presentinvention are not limited to adenocarcinoma, squamous cell carcinoma,small cell carcinoma, and large cell carcinoma, and may be derived fromany tissue type including sarcoma. For example, cancers which can betreated by the pharmaceutical composition for treating cancer or themedicament for treating cancer of the present invention include, but arenot limited to, lung cancer, gastric cancer, esophageal cancer, livercancer, biliary tract cancer, pancreatic cancer, large bowel cancer,renal cancer, bladder cancer, prostatic cancer, testicular cancer,uterine cancer, ovarian cancer, breast cancer, skin cancer, laryngealcancer, colorectal cancer, melanoma, thyroid cancer, fibrosarcoma, skinfibrosarcoma, uterine sarcoma, liposarcoma, myosarcoma, hemangiosarcoma,Kaposi's sarcoma, lymphangiosarcoma, osteosarcoma, leukemia, lymphoma,myeloma, and the like.

The pharmaceutical composition for treating cancer or the medicament fortreating cancer of the present invention may contain additives which canbe used in medicaments, such as pharmaceutically acceptable carriers,diluents, excipients, and stabilizers. These additives can beappropriately selected based on the common technical knowledge of thoseskilled in the art.

The cell extract component having cytocidal activity derived frommalignant tumor-derived cells, which is obtained by the above-describedpreparation method of the present invention, is expected to be awater-soluble low molecular weight compound having a molecular weight of1 kDa or less, and thus is expected to be used in various forms as amedicament. For example, the pharmaceutical composition for treatingcancer or the medicament for treating cancer of the present inventioncan be administered to a subject orally or parenterally such as byinjection.

EXAMPLES

Hereinafter, the present invention is described in more detail withreference to specific examples, but the scope of the present inventionis not limited to these examples.

[Malignant Tumor-Derived Established Cell Lines Used]

(1) Human Renal Cancer-Derived Cell Line: HRC23

HRC23 is one established as a cell line by transplantation of humanrenal cell carcinoma in nude mice. For passage, phenol red- andantibiotic-free Eagle's MEM (Nissui Pharmaceutical Co., Ltd.)supplemented with 10% FBS was used, and for cell detachment, 0.1%trypsin and 0.01% EDTA were used. In addition, cloning was carried outas needed. The culturing was carried out at 37° C. under 5% CO₂.

(2) Mouse Lewis Lung Carcinoma-Derived Cell Line

A cell line distributed by RIKEN was used (RCB0558: LLC). For passage,Eagle's MEM supplemented with 10% FBS was used, and for cell detachment,0.1% trypsin and 0.01% EDTA were used. The culturing was carried out at37° C. under 5% CO₂. The cells are highly metastatic and resistant tovarious anticancer agents, and are cells obtained by culturing oneswhich have been passaged in vivo (Bertram J S, Janik P., Cancer Lett.1980 November, 11(1), p. 63-73).

(3) Other Human Malignant Tumor-Derived Cell Lines

Four types of cell lines derived from human malignant tumors were used.All of these cells were obtained from the National Institute forBiomedical Innovation, the Japanese Collection of Research BioresourcesCell Bank. For passage and assay, the indicated media and celldetachment enzymes were used. The culturing was carried out at 37° C.under 5% CO₂.

Table 1 presents the types of cells used.

TABLE 1 Derived Tissue Culture Medium Cell Name From Type (+10% FBS)HRC23 Human Renal Cell Adenocarcinoma Eagle's MEM Carcinoma MKN74 HumanGastric Adenocarcinoma RPMI1640 (JCRB0255) Cancer LK-2 Human LungSquamous Cell RPMI1640 (JCRB0829) Cancer Carcinoma VMRC-JCP Human LungSquamous Cell RPMI1640 (JCRB0103) Cancer Carcinoma SKN Human UterineLeiomyosarcoma Eagle's MEM (JCRB0173) Sarcoma LLC Mouse Lewis LungMedullary Eagle's MEM (RCB0558) Carcinoma Carcinoma

Pathologically, malignant tumors are roughly classified into epithelialand non-epithelial ones, and many of them are epithelial. In Table 1,SKN is non-epithelial, and the others are epithelial, where LLC is knownas a mouse-derived epithelial malignant tumor cell which is different inspecies and resistant to various anticancer agents. In addition,representative cells were selected in terms of tissue type.

Example 1: Preparation of Undiluted Solution Sample

(1) Undiluted Solution Sample Prepared Using Serum-Containing Medium

HRC23 was cultured in Eagle's MEM with 10% FBS in the same manner asthat of passage. The Eagle's MEM used was an antibiotic- and phenolred-free medium. HRC23 was cultured in a flask until cell growth reacheda confluent state, and was further cultured to overgrowth(overpopulation). Then, the medium was finally replaced with Eagle's MEMwith 10% FBS as described above, followed by incubation at 37° C. under5% CO₂. After nine days, death of the cells was observed inmorphological aspect of HRC23. After that, the medium was recovered andcentrifuged at 3×10³×g for 10 minutes to obtain a supernatant. Thissupernatant was filtered with a 0.1 μm membrane filter (Millex® VV,Merck & Co., Inc. (Millipore)), and the filtrate was stored asepticallyat 4° C. as an undiluted solution sample.

(2) Undiluted Solution Sample Prepared Using Serum-Free Medium

HRC23 was cultured in Eagle's MEM with 10% FBS in the same manner asthat of passage. The Eagle's MEM used was an antibiotic- and phenolred-free medium. HRC23 was cultured in a flask until cell growth reacheda confluent state, and was further cultured to overgrowth(overpopulation). Then, the medium was replaced with serum-free Eagle'sMEM not containing antibiotics and phenol red, followed by incubation at37° C. under 5% CO₂ for 5 to 7 hours, during which the cells were rinsedseveral times with the same serum-free Eagle's MEM as above. The mediumwas finally replaced with the same serum-free Eagle's MEM as above,followed by incubation at 37° C. under 5% CO₂. After nine days, death ofthe cells was observed in morphological aspect of HRC23. After that, themedium was recovered and centrifuged at 3×10³×g for 10 minutes to obtaina supernatant. This supernatant was filtered with a 0.1 μm membranefilter (Millex® VV, Merck & Co., Inc. (Millipore)), and the filtrate wasstored aseptically at 4° C. as an undiluted solution sample.

In addition, this undiluted solution sample was ultrafiltered to collecta fraction with a molecular weight of 1 kDa or less (Stirred Cell Model8050 equipped with Ultracel® Amicon® YM1 and Ultracel® ultrafiltrationmembrane PLAC04310, Merck & Co., Inc. (Millipore)), and the resultantone was stored aseptically at 4° C.

(3) Undiluted Solution Sample Prepared Using Physiological Buffer SaltSolution

HRC23 was cultured in Eagle's MEM with 10% FBS in the same manner asthat of passage. The Eagle's MEM used was an antibiotic- and phenolred-free medium. HRC23 was cultured in a flask until cell growth reacheda confluent state, and was further cultured to overgrowth(overpopulation). Then, the medium was replaced with Hanks' balancedsalt solution without antibiotics and glucose (pH 7.3, also referred toas HBSS−), followed by incubation at 37° C. under 5% CO₂ for 5 to 7hours, during which the cells were rinsed several times with the sameHanks' balanced salt solution as above. After that, the cells wereincubated at 37° C. under 5% CO₂ in the final Hanks' balanced saltsolution which is the same as that described as above. After four days,death of the cells was observed in morphological aspect of HRC23. Afterthat, the Hanks' balanced salt solution was recovered and centrifuged at2×10³×g for 10 minutes to obtain a supernatant. This supernatant wasfiltered with a 0.1 μm membrane filter (Millex® VV, Merck & Co., Inc.(Millipore)), and the filtrate was stored aseptically at 4° C. as anundiluted solution sample.

In addition, this undiluted solution sample was ultrafiltered to collecta fraction with a molecular weight of 1 kDa or less (Stirred Cell Model8050 equipped with Ultracel® Amicon® YM1 and Ultracel® ultrafiltrationmembrane PLAC04310, Merck & Co., Inc. (Millipore)), and the resultantone was stored aseptically at 4° C.

Moreover, instead of glucose-free Hanks' balanced salt solution (HBSS−),glucose-free Earle's balanced salt solution (Earle) and glucose-freephosphate buffered saline (PBS(+)) were used to prepare undilutedsolution samples. Table 2 shows the compositions of the glucose-freeHanks' balanced salt solution, the Earle's balanced salt solution, andthe phosphate buffered saline.

TABLE 2 HBSS- Earle PBS(+) (DPBS) [g/L] [g/L] [g/L] NaCl 8.00 6.80 8.00KCl 0.40 0.40 2.00 CaCl₂ 0.14 0.20 0.10 MgSO₄ 0.098 0.10 MgCl₂•12H₂O0.10 NaH₂PO₄ 0.125 Na₂HPO₄•12H₂O 0.139 2.90 KH₂PO₄ 0.06 0.20 Glucose NotContained Not Contained Not Contained NaHCO₃ 0.35 2.20 Vapor Layer air5% CO₂ air

Example 2: Test for Cytocidal Activity

(1) Preparation of Serial Dilution Series of Test Samples

To the undiluted solution sample (serum-containing medium) prepared inExample 1 (1) above, 10% FBS, an amino acid-blended solution and avitamin-blended solution for Eagle's MEM (KOHJIN BIO; the aminoacid-blended solution was a 50 times concentrated solution, and thevitamin-blended solution was a 100 times concentrated solution), andglucose were newly added in amounts prescribed for Eagle's MEM. The pHof the resultant solution was adjusted to 7.1 to 7.4 with 7.5% NaHCO₃,and then a prescribed amount of glutamine and 10% volume of FBS wereadded to the resultant solution to prepare a test sample. This is forthe purpose of supplementing the nutrients consumed after the finalreplacement of medium with serum-containing medium. This test sample wassubjected to 2-fold serial dilution with a control medium (Eagle's MEMwith 10% FBS) to prepare a serial dilution series of test samples.

To the undiluted solution sample (serum-free medium) prepared in Example1 (2) above, an amino acid and a vitamin were added as in the case ofthe undiluted solution sample of the serum-containing medium. The pH ofthe resultant solution was adjusted, and then glutamine, 10% FBS, andglucose were added to the resultant solution to prepare a test sample.This test sample was subjected to 2-fold serial dilution with a controlmedium (Eagle's MEM with 10% FBS) to prepare a serial dilution series oftest samples. In addition, for the undiluted solution sample, which wassubjected to ultrafiltration to collect a fraction with a molecularweight of 1 kDa or less, a serial dilution series of test samples wasprepared in the same manner.

To the undiluted solution sample (Hanks' balanced salt solution)prepared in Example 1 (3) above, an amino acid and a vitamin were addedas in the case of the sample undiluted solution of the serum-containingmedium. The pH of the resultant solution was adjusted, and thenglutamine, 10% FBS, and glucose were added to the resultant solution toprepare a test sample. This test sample was subjected to 2-fold serialdilution with a control solution (Eagle's MEM with 10% FBS) to prepare aserial dilution series of test samples. In addition, for the undilutedsolution sample, which was subjected to ultrafiltration to collect afraction with a molecular weight of 1 kDa or less, a serial dilutionseries of test samples was prepared in the same manner.

(2) Measurement of Cell Survival Rate by MTT Assay

HRC23 was diluted at a dilution rate for ordinary passage, dispensedinto a 96-well microplate, and cultured in Eagle's MEM with 10% FBS at37° C. under 5% CO₂. After 24 hours (Day 1), the medium was replacedwith 170 μL of each serially diluted test sample, and moreover wasincubated with replacement twice with each fresh test sample every otherday (Day 3 and Day 5). On Day 6, the cell survival rate was measured byMTT assay (n=3).

The MTT assay was carried out as follows. MTT (Dojindo) was dissolved inDulbecco's phosphate buffered saline (PBS−) without calcium andmagnesium to a concentration of 5 mg/mL that is a 10-fold concentratedsolution. This was aseptically filtered with a 0.1 μm membrane filter,dispensed, and stored at 4° C. After the cells were washed with 200 μLof medium (Eagle's MEM with 10% FBS), the above-described 5 mg/mL MTTsolution is added to each medium in an amount of 1/10 volume of themedium to obtain a solution containing 0.5 mM MTT, and 150 μL thereofwas added to each well of the 96-well microplate. After incubation for30 to 40 minutes at 37° C., the solution in each well was aspirated offand washed with 200 μL of the medium. Then, 200 μL of dimethyl sulfoxidewas added to each well. The absorbance at a wavelength of 570 nm wasmeasured with a microplate reader (Model 550, Bio-Rad Laboratories). Thecell survival rate was calculated by the following formula.

$\begin{matrix}{{{Cell}\mspace{14mu}{Survival}\mspace{14mu}{Rate}\mspace{14mu}(\%)} = {\frac{( {{A\mspace{14mu}{sample}} - {A\mspace{14mu}{blank}}} )}{( {{A\mspace{14mu}{control}} - {A\mspace{14mu}{blank}}} )} \times 100}} & \lbrack {{Formula}\mspace{14mu} 1} \rbrack\end{matrix}$

A_(sample) denotes the absorbance measured as described above using eachof the serially diluted test samples.

A_(control) denotes the absorbance measured as described above butwithout using each of the serially diluted test samples.

A_(blank) denotes the absorbance measured as described above but withoutHRC23.

FIGS. 1 to 3 present the results. A concentration-dependent cytocidalactivity was observed in any of the sample prepared using theserum-containing medium, the sample prepared using the serum-freemedium, and the sample prepared using the physiological buffered saltsolution. In addition, a concentration-dependent cytocidal activity wassimilarly observed in the fraction with a molecular weight of 1 kDa orless of the sample prepared using the serum-free medium and the fractionwith a molecular weight of 1 kDa or less of the sample prepared usingthe physiological buffer salt solution.

The sample prepared using physiological buffer salt solution is oneprepared using glucose-free Hanks' balanced salt solution, that is, amedium without nutrients and energy sources. To put it differently, thisindicates that the cells produced a substance having cytocidal activity,as in the case of the presence of nutrients and energy sources, eventhough the supply of external nutrients and energy sources was blocked.This is a surprising result. It has been revealed from the result thatcells produce a substance having cytocidal activity using only thesubstances present in the cells as materials, and cause themselves todie.

Example 3: Preparation of Cell Extract Component Having CytocidalActivity

(1) Concentration

For large scale culture, an undiluted solution sample was prepared usinga flask with a surface area of 181 cm² in the same manner as the case ofpreparing the undiluted solution sample in Example 1 (3) above.Glucose-free Hanks' balanced salt solution (HBSS−) was used as aphysiological buffer salt solution, and 40 mL of HBSS− was used in thefinal physiological buffer salt solution replacement. The undilutedsolution sample thus obtained was evaporated to dryness under reducedpressure, and ethanol was added to a dried residue in an amount of 1/10volume of the undiluted solution ample to dissolve the dried residue.Centrifugation was carried out at 3×10³×g for 10 minutes to obtain asupernatant, followed by evaporation to dryness under reduced pressureagain. This ethanol extraction was repeated to obtain a dried productconcentrated to about 1000 times of the undiluted solution sample. Thisdried product was stored at −80° C.

(2) Gel Filtration Chromatography

The dried product obtained in Example 3 (1) above was dissolved in purewater and washed with a liquid mixture of chloroform and ethyl acetate,from which the aqueous layer was recovered to obtain a sample. Thewashed sample was dissolved in 300 μL of 50 mM Na₂SO₄, and gelfiltration chromatography was carried out with the following apparatusand conditions.

Liquid feed pump: 880 PU (JASCO Corporation)

Detector: 825 UV (JASCO Corporation)

Mixer: HG-980-31 (JASCO Corporation)

Injector: Rheodyne 8125 (Rheodyne)

Column: Superformance (26 mm×600 mm) (Merck & Co., Inc.)

Carrier: HP Cellulofine sf (CHISSO CORPORATION)

Mobile phase: 50 mM Na₂SO₄

Flow rate: 0.6 ml/min

Fraction size: 1.8 ml (3 min)

Detection: 230 nm; Sensitivity: 0.16 aufs

FIG. 4 presents the results of gel filtration chromatography. The eightfractions A to H illustrated in FIG. 4 were collected, and cytocidalactivity was measured for each fraction by MTT assay. As a result,cytocidal activity was detected only in the fraction A (105 to 141 min).

The MTT assay here was carried out as follows. HRC23 was diluted at adilution rate for ordinary passage, dispensed into a 96-well microplate,and cultured in Eagle's MEM with 10% FBS at 37° C. under 5% CO₂. After24 hours, the medium was replaced with 170 μL of sample prepared fromeach fraction, followed by incubation for 2 days. Then, 200 μL ofdimethyl sulfoxide was added to each well. The absorbance at awavelength of 570 nm was measured with a microplate reader (Model 550,Bio-Rad Laboratories). The sample was prepared as follows. The aliquotof each fraction was added with the same volume of methanol, followed byfiltration through a 0.22 μm membrane filter (Millex® GV, Merck & Co.,Inc. (Millipore)) to remove Na₂SO₄. After that, a dried product obtainedby drying the filtrate was dissolved in Eagle's MEM with 10% FBS andsubjected to 2-fold serial dilution.

FIG. 5 illustrates the appearance of color development on a microplateobserved by MTT assay, together with a graph illustrating theconcentration dependence of the cell survival rate. In FIG. 5, thesample concentration is in terms of undiluted solution.

The fraction A recovered was added with the same volume of methanol,followed by filtration through a 0.22 μm membrane filter (Millex® GV,Merck & Co., Inc. (Millipore)) to remove Na₂SO₄. After that, thefiltrate was evaporated to dryness under reduced pressure, and theresultant dried product was stored at −80° C.

(3) Ion Exchange Chromatography

The dried product obtained in Example 3 (2) above was dissolved in 200μL of 0.15 M Na₂SO₄, which was used as a sample to carry out ionexchange chromatography with the following apparatus and conditions. Theactive fractions were separated using the linear concentration gradientmethod of Na₂SO₄ on a strong cation exchange resin.

Liquid feed pump: 880 PU (JASCO Corporation)

Detector: 825 UV (JASCO Corporation)

Mixer: HG-980-31 (JASCO Corporation)

Injector: Rheodyne 8125 (Rheodyne)

Column: Resource™ S; 1 ml (GE Healthcare), two columns were used inseries

Elution was carried out by the linear concentration gradient method asshown in Table 3.

Flow rate: 0.25 ml/min

Fraction size: 0.75 ml (3 min)

Mobile phase: A; H₂O, B; 0.3M Na₂SO₄

Detection: 230 nm; Sensitivity: 0.16 aufs

TABLE 3 Elution Program Time (Min) 0 30 150 151 Mobile Phase A (%) 50 5020 1 Mobile Phase B (%) 50 50 80 99

The sample was injected into a column previously equilibrated with 0.15M Na₂SO₄ solution, followed by washing with the solution for 30 minutes.After that, elution was carried out by the linear concentration gradientmethod of 0.15 M to 0.24 M Na₂SO₄.

FIG. 6 presents the results of ion exchange chromatography. Cytocidalactivity was measured for each fraction by MTT assay, and cytocidalactivity was detected only in fractions 18 to 21 having a Na₂SO₄concentration of 165 to 170 mM. The MTT assay here was carried out inthe same manner as in Example 3 (2) above.

Each of the recovered fractions having a Na₂SO₄ concentration of 165 to170 mM was added with the same volume of methanol, followed byfiltration through a 0.22 μm membrane filter (Millex® GV, Merck & Co.,Inc. (Millipore)) to remove Na₂SO₄. After that, the filtrate wasevaporated to dryness under reduced pressure, and the resultant driedproduct was stored at −80° C.

(4) Re-Gel Filtration Chromatography

The dried product obtained in Example 3 (3) above was dissolved in 300μL of 50 mM Na₂SO₄, which was used as a sample to carry out gelfiltration chromatography again with the following apparatus andconditions.

Liquid feed pump: 880 PU (JASCO Corporation)

Detector: 825 UV (JASCO Corporation)

Mixer: HG-980-31 (JASCO Corporation)

Injector: Rheodyne 8125 (Rheodyne)

Column: Superformance (26 mm×600 mm) (Merck & Co., Inc.)

Carrier: HP Cellulofine sf (CHISSO CORPORATION)

Mobile phase: 50 mM Na₂SO₄

Flow rate: 0.6 ml/min

Fraction size: 1.2 ml (2 min)

Detection: 205 nm; Sensitivity: 0.16 aufs

FIG. 7 presents the results of gel filtration chromatography. Cytocidalactivity was measured for each fraction by MTT assay, and cytocidalactivity was observed in three fractions of fraction 57 (elution time112 to 114 min), fraction 58 (elution time 114 to 116 min), and fraction59 (elution time 116 to 118 min) in the earlier absorption peak. Thecytocidal activity was strongly observed especially in fraction 58. TheMTT assay here was carried out in the same manner as in Example 3 (2)above.

Each of the three recovered fractions was added with the same volume ofmethanol, followed by filtration through a 0.22 μm membrane filter(Millex® GV, Merck & Co., Inc. (Millipore)) to remove Na₂SO₄. Afterthat, the filtrate was evaporated to dryness under reduced pressure toobtain a dried product.

(5) Mass Spectrometry (TOF-MS)

The dried product obtained in Example 3 (3) above was resuspended in 10μL of a buffer solution composed of 5 mg/mL α-cyano-4-hydroxycinnamicacid (α-CHCA) in acetonitrile:water:TFA (50:50:1), which was used as asample and analyzed by mass spectrometry with the following apparatusand conditions.

Analysis was carried out according to the peptide analysis program usingthe following.

Analytical instrument: Voyager System 6366 (Applied Biosystem)

Applied voltage: +20000 V

Sample introduction: manual; MALDI plate

Matrix: α-Cyano-4-hydroxycinnamic acid (α-CHCA) (Tokyo Chemical IndustryCo., Ltd.)

FIGS. 8 to 10 present the results obtained from the three fractions (57,58, and 59) with m/z values of 100 to 1500. In addition, FIG. 11presents the results obtained from the matrix alone with m/z values of100 to 1000.

In each of the mass spectra for the three fractions (57, 58, and 59),signals were observed at m/z values of 600 and 714. In particular,strong signals were observed at m/z values of 600.16 and 714.04 in thespectrum of fraction 58, while the signals were not as clear in thespectrum of fraction 59 as in fractions 57 and 58. In addition, in thespectrum of fraction 58, a signal was clearly observed at m/z value of114.09 (an enlarged diagram is illustrated as FIG. 12).

Meanwhile, no signal having an m/z value greater than the m/z value of1000 was observed. Therefore, it is suggested that the cell extractcomponent having cytocidal activity has a molecular weight of 1 kD orless. This is consistent with the results of FIGS. 2 and 3 indicatingthat cytocidal activity was observed in a fraction having a molecularweight of 1 kDa or less.

However, in light of the fact that there are many signals having m/zvalues of 300 or less also in the mass spectra obtained as describedabove, the identification of a cell extract component having cytocidalactivity is considered to require further detailed examination includingexamination on the stability of the component.

Example 4: Measurement of Cytocidal Activity Against Various CancerCells

The dried product obtained in Example 3 (2) above was subjected to2-fold serial dilution using the culture media used for culturing HRC23,MKN74, LK2, VMRC-JCP, SKN, and LLC to prepare a serial dilution seriesof test samples. Each of HRC23, MKN74, LK2, VMRC-JCP, SKN, and LLC wasseparately seeded in a 96-well microplate in an amount for establishingan approximately 80% confluent state in 3 days of culture, and wascultured for 24 hours at 37° C. under 5% CO₂ in the predeterminedculture medium. The medium was replaced with 170 μL of test sample,followed by incubation for 2 days. Then, 200 μL of dimethyl sulfoxidewas added to each well. The absorbance at a wavelength of 570 nm wasmeasured with a microplate reader (Model 550, Bio-Rad Laboratories) andMTT assay was carried out (n=3).

FIG. 13 illustrates a graph of cell survival rate of various cancercells measured by MTT assay. The horizontal axis is represented on acommon logarithmic scale, where the minimum concentration of the testsample exhibiting 0% survival rate for HRC23 is expressed as 1 inarbitrary unit. The results have revealed that the test sample havingcytocidal activity obtained from HRC23 is also effective against cancersother than HRC23, and commonly exhibits cytocidal activity in aconcentration-dependent manner regardless of the type of cancer andtissue type.

Example 5: Measurement of Cytocidal Activity in LLC- or SKN-DerivedSample

(1) Preparation of Undiluted Solution Sample

LLC and SKN were cultured in Eagle's MEM with 10% FBS in the same manneras that of HRC23. The Eagle's MEM used was an antibiotic- and phenolred-free medium. LLC and SKN were cultured in a flask until their cellgrowth reached a confluent state, and were further cultured for one day.After washing with Hanks' balanced salt solution without antibiotics andglucose, incubation was carried out at 37° C. under 5% CO₂ in 5 mLHanks' balanced salt solution/flask. After death of LLC and SKN had beenobserved in morphological aspect of the cells, Hanks' balanced saltsolution was recovered and centrifuged at 1,500×g for 10 minutes toobtain supernatants. These supernatants were filtered with a 0.1 μmmembrane filter (Millex® VV, Merck & Co., Inc. (Millipore)), and thefiltrates were each used as an undiluted solution sample.

In addition, each of these undiluted solution samples was ultrafilteredto collect a fraction with a molecular weight of 1 kDa or less (StirredCell Model 8050 equipped with Ultracel® Amicon® YM1 and Ultracel®ultrafiltration membrane PLAC04310, Merck & Co., Inc. (Millipore)).

(2) Measurement of Cell Survival Rate by MTT Assay

To the undiluted solution sample prepared in Example 5 (1) above, 10%FBS, amino acid- and vitamin-blended solutions for Eagle's MEM, andglucose were added in amounts prescribed for Eagle's MEM. The pH of theresultant solution was adjusted with 7.5% NaHCO₃ to prepare a testsample. This test sample was subjected to 2-fold serial dilution with acontrol solution (Hanks' balanced salt solution with 50-foldconcentration amino acid-blended solution and a 100-fold concentrationvitamin-blended solution for Eagle's MEM) to prepare a serial dilutionseries of test samples. In addition, the undiluted solution sample wassubjected to ultrafiltration to collect a fraction with a molecularweight of 1 kDa or less, for which a serial dilution series was preparedin the same manner.

MTT assay was carried out according to the method described in Example 2(2) above to measure the HRC23 cell survival rate (n=3). FIGS. 14 and 15present the results.

Both the LLC-derived sample and the SKN-derived sample also exhibitedcytocidal activity against HRC23 in the same manner as the HRC23-derivedsample. The result that the LLC-derived sample exhibited cytocidalactivity against HRC23 and the result in Example 4 (the HRC23-derivedsample exhibited cytocidal activity against LLC) mean that crossreactions across species were observed. In addition, SKN is a rarenon-epithelial malignant tumor cell, and it has been confirmed thatnon-epithelial malignant tumor cells such as SKN also produce asubstance having cytocidal activity. As for the SKN-derived sample, thecytocidal activity observed was weaker than that of the LLC-derivedsample and the HRC23-derived sample. This seems to be because the numberof SKN cells after culturing was much smaller than that of LLC or HRC23.

Example 6: Measurement of Cytocidal Activity when Using VariousPhysiological Buffer Salt Solutions

To the undiluted solution samples prepared in Example 1 (3) above(HBSS−, Earle, and PBS(+)), 10% FBS, 50-fold concentration amino acid-and 100-fold concentration vitamin-blended solutions for Eagle's MEM,and glucose were added in amounts prescribed for Eagle's MEM. The pH ofeach resultant solution was adjusted with 7.5% NaHCO₃ to prepare testsamples. Each of these test samples was subjected to 2-fold serialdilution with a control solution (HBSS−, Earle, or PBS(+) with 50-foldconcentration amino acid-blended solution and a 100-fold concentrationvitamin-blended solution for Eagle's MEM) to prepare a serial dilutionseries of test samples.

MTT assay was carried out according to the method described in Example 2(2) above to measure the HRC23 cell survival rate (n=3). FIG. 16presents the results.

Cytocidal activity was observed with different intensities in each ofthe case using HBSS−, the case using Earle, and the case using PBS(+).

Example 7: Evaluation of Influence of Cell Culture States on CytocidalActivity

In the case of using an ordinary culture medium, since cells in thegrowth phase continue to grow, tests with different cell densities inthe culture medium are difficult. However, use of Hanks' balanced saltsolution containing no nutrient sources (glucose free) has made itpossible to investigate the production of a component having cytocidalactivity at the stage of growth phase.

HRC23 was seeded in 25 cm² passage culture flasks with different numbersof cells, and they were cultured in Eagle's MEM with 10% FBS. TheEagle's MEM used was an antibiotic- and phenol red-free medium. Thecells were cultured in the flask with a large number of cells seededuntil the cell growth reached a confluent state, and further culturedfor one day. At this point in time, the cells in the flask with a smallnumber of cells seeded was in the growth phase and the cell density wasapproximately 72% compared to the confluent state although the celldensity was a level that does not pose an obstacle for passage. At thispoint in time, in any of the case of a large number of cells seeded andthe case of a small number of cells seeded, the cells were washed withHanks' balanced salt solution without antibiotics and glucose and thenincubated at 37° C. under 5% CO₂ in 5 mL Hanks' balanced saltsolution/flask. After death of the cells was observed in morphologicalaspect of HRC23, the Hanks' balanced salt solution was recovered andcentrifuged at 1,500×g for 10 minutes to obtain a supernatant. Thissupernatant was filtered with a 0.1 μm membrane filter (Millex® VV,Merck & Co., Inc. (Millipore)), and the filtrate was used as anundiluted solution sample.

MTT assay was carried out (in a 25 cm² passage culture flask) accordingto the method described in Example 2 (2) above to measure the HRC23 cellsurvival rate (n=3). FIG. 17 presents the results.

Cytocidal activity was observed with different intensities in each ofthe case of a large number of cells seeded and the case of a smallnumber of cells seeded. Therefore, it has been revealed that even cellsin the growth phase produce a component having cytocidal activity. Thisindicates that cells can intentionally produce a component having acytocidal effect regardless of the phase of the cells.

Example 8: Measurement of Cytocidal Activity of LK-2-Derived SampleAgainst LK-2 and HRC23

(1) Preparation of Undiluted Solution Sample

LK-2 was cultured in RPMI 1640 with 10% FBS (Merck & Co., Inc.(Sigma-Aldrich Japan), R883) in the same manner as that of passage. LK-2was cultured in a flask until cell growth reached a fully confluentstate, and then the medium was finally replaced with 5 mL of RPMI 1640with 10% FBS. In addition, apart from the above-described example ofreplacing the medium with RPMI 1640 with 10% FBS, LK-2 was cultured in aflask until cell growth reached a fully confluent state, and the cellswere washed 4 times with 30 mL of Hanks' balanced salt solution withoutantibiotics and glucose, and then 5 mL of the Hanks' balanced saltsolution was placed in a flask to immerse the cells.

The above two types of culture solutions were incubated at 37° C. under5% CO₂, and the cells were cultured until death of the cells had beenobserved in morphological aspect of LK-2. After that, the medium andHanks' balanced salt solution were recovered and centrifuged at 1,500×gfor 10 minutes to obtain supernatants. These supernatants were filteredwith a 0.1 μm membrane filter (Millex® VV, Merck & Co., Inc.(Millipore)), and the filtrates were each used as an undiluted solutionsample. In addition, the undiluted solution sample obtained by usingHanks' balanced salt solution was ultrafiltered to collect a fractionwith a molecular weight of 1 kDa or less (Stirred Cell Model 8050equipped with Ultracel® Amicon® YM1 and Ultracel® ultrafiltrationmembrane PLAC04310, Merck & Co., Inc. (Millipore)).

(2) Preparation of Serial Dilution Series of Test Samples

To each of the undiluted solution samples prepared in Example 8 (1)above, an amino acid-blended solution (50-fold concentrated) (Merck &Co., Inc. (Sigma-Aldrich Japan), M5550) and a vitamin-blended solution(100-fold concentrated) (Merck & Co., Inc. (Sigma-Aldrich Japan), R7256)for RPMI 1640 were added. The pH of each resultant solution was adjustedto 7.2 to 7.3 with acetic acid, and then 10% FBS, 10% glucose, and 0.1volume of 200 mM glutamine were added to each resultant solution toprepare test samples. Each test sample was subjected to 2-fold serialdilution in a control medium (RPMI 1640 with 10% FBS) to prepare aserial dilution series A of test samples. In addition, the undilutedsolution sample was subjected to ultrafiltration to collect a fractionwith a molecular weight of 1 kDa or less, for which a serial dilutionseries A (<1 kDa) was prepared in the same manner.

In addition, to each of the undiluted solution samples prepared inExample 8 (1) above, an amino acid-blended solution (50-foldconcentrated) (same as above) and a vitamin-blended solution (100-foldconcentrated) (same as above) for Eagle's MEM were added. The pH of eachresultant solution was adjusted to 7.2 to 7.3 with acetic acid, and then10% FBS, 10% glucose, and 0.1 volume of 200 mM glutamine were added toeach resultant solution to prepare test samples. Each test sample wassubjected to 2-fold serial dilution in a control medium (Eagle's MEMwith 10% FBS) to prepare a serial dilution series B of test samples. Inaddition, the undiluted solution sample was subjected to ultrafiltrationto collect a fraction with a molecular weight of 1 kDa or less, forwhich a serial dilution series B (<1 kDa) was prepared in the samemanner.

(3) Measurement of Cell Survival Rate by MTT Assay

LK-2 was diluted at a dilution rate for ordinary passage, dispensed intoa 96-well microplate, and cultured in RPMI 1640 with 10% FBS at 37° C.under 5% CO₂ for 24 hours. Then, each medium was replaced with 170 μL ofa diluted solution of the serial dilution series A. After culturing for24 hours in the diluted solutions of the serial dilution series A, eachdiluted solution was replaced with a fresh diluted solution of theserial dilution series A at the same dilution factor, and the culturingwas carried out for another 24 hours. Also in the case of using theserial dilution series A (<1 kDa), LK-2 was cultured as in the case ofusing the serial dilution series A.

MTT assay was carried out according to the method described in Example 2(2) above to measure the LK-2 cell survival rate (n=3). FIG. 18 presentsthe results.

In addition, HRC23 was diluted at a dilution rate for ordinary passage,dispensed into a 96-well microplate, and cultured in Eagle's MEM with10% FBS at 37° C. under 5% CO₂ for 24 hours. Then, each medium wasreplaced with 170 μL of a diluted solution of the serial dilution seriesB. After culturing for 24 hours in the diluted solutions of the serialdilution series B, each diluted solution was replaced with a freshdiluted solution of the serial dilution series B at the same dilutionfactor, and the culturing was carried out for another 24 hours. Also inthe case of using the serial dilution series B (<1 kDa), HRC23 wascultured as in the case of using the serial dilution series B.

MTT assay was carried out according to the method described in Example 2(2) above to measure the HRC23 cell survival rate (n=3). FIG. 19presents the results.

The LK-2-derived sample exhibited cytocidal activity against LK-2 andHRC23. In addition, the sample based on the undiluted solution sampleobtained using Hanks' balanced salt solution exhibited strongercytocidal activity than the sample based on the undiluted solutionsample obtained by finally replacing the medium with RPMI 1640. Thissuggests that obtaining a component having cytocidal activity extractedfrom malignant tumor-derived cells using a physiological buffer saltsolution such as Hanks' balanced salt solution may make it possible toachieve higher yields than using a culture medium to obtain thecomponent, or may make it possible to obtain the target componentwithout reducing cytocidal activity of the component.

Example 9: In Vivo Cytocidal Activity on Mice

Four mice (C57BL/6NCrSIc, male, 5 weeks of age) were inoculatedintraperitoneally with 300 μL of LLC suspension (2×10⁶ cells) for LLCtransplantation. After 1 week, the dried product obtained in the above[4] (2) equivalent to 1 L of undiluted solution was dissolved in 300 μLof FBS-free Eagle's MEM, and the resultant solution wasintraperitoneally administered to two mice as a treatment group once aday for 6 days. The two mice in the control group, not subjected to theabove treatment, died 25 days after LLC transplantation. On the otherhand, in the treatment group, one died on day 35 and the other died onday 48 after LLC transplantation. A clear survival benefit was observedin the treatment group as compared with the control group. In thetreatment group, no symptom that appeared to be a side effect wasobserved.

What is claimed is:
 1. A method for producing a physiological buffersalt solution comprising a cytocidal component produced by malignanttumor cells, the method comprising: culturing malignant tumor cellsobtained from a malignant tumor or from an established malignant tumorcell line in a culture medium to a 60% to 100% confluent state;replacing, after the culturing, the culture medium with a physiologicalbuffer salt solution; and recovering the physiological buffer saltsolution after the time at which death of the malignant tumor cells isobserved in the physiological buffer salt solution in morphologicalaspect of said cells, wherein the recovered physiological buffer saltsolution comprises a cytocidal component produced by the malignant tumorcells, wherein the physiological buffer salt solution is glucose-free.2. The production method according to claim 1, wherein the physiologicalbuffer salt solution is selected from the group consisting of Hanks'balanced salt solution, Earle's balanced salt solution, and phosphatebuffered saline.
 3. A method for preparing a cell extract cytocidalcomponent isolated from or purified from malignant tumor cells, themethod comprising: culturing malignant tumor cells obtained from amalignant tumor or from an established malignant tumor cell line in aculture medium to a 60% to 100% confluent state; replacing, after theculturing, the culture medium with a physiological buffer salt solution;recovering the physiological buffer salt solution after the time atwhich death of the malignant tumor cells is observed in thephysiological buffer salt solution in morphological aspect of saidcells; drying the recovered physiological buffer salt solution to obtaina dried product of the recovered physiological buffer salt solution,wherein the dried product contains a fraction with a molecular weight of1 kDa or less in the recovered physiological buffer salt solution;extracting the dried product using a solvent containing an alcoholhaving 1 to 3 carbon atoms, and drying a resultant solution; dissolvinga dried product of the resultant solution in water to obtain an aqueoussolution, adding a non-polar organic solvent to the aqueous solution toform an aqueous layer and an organic layer, and extracting the aqueouslayer; and separating a cell extract cytocidal component bychromatography of the extracted aqueous layer, wherein the physiologicalbuffer salt solution is glucose-free.
 4. The preparation methodaccording to claim 3, wherein the chromatography includes gel filtrationchromatography and/or cation exchange chromatography.
 5. The productionmethod according to claim 1, wherein the malignant tumor cells are notgenetically engineered.
 6. The production method according to claim 3,wherein the physiological buffer salt solution is selected from thegroup consisting of Hanks' balanced salt solution, Earle's balanced saltsolution, and phosphate buffered saline.
 7. The production methodaccording to claim 1, wherein the malignant tumor cells are notgenetically engineered.
 8. The production method according to claim 2,wherein the malignant tumor cells are not genetically engineered.
 9. Thepreparation method according to claim 3, wherein the malignant tumorcells are not genetically engineered.